The measurement of thyroid hormones in biological fluids has been the object of intense research for a number of years. Proposed methods include determination of bound as well as unbound thyroid hormones. Although the major portion (&gt;99%) of thyroid hormones found in biological fluids are bound to endogenous binding proteins such as thyroxin-binding globulin (TBG) and thyroxin-binding pre-albumin (TBPA) in serum, the determination of free (unbound) thyroid hormones as a measure of thyroid function has attracted the most attention. Considered critically important because of its physiological significance, efforts have been made to estimate the concentration of free thyroxine hormones by a variety of so-called direct and indirect methods using radioimmunoassay (RIA) as the principal measurement system. Nevertheless, attempts to increase the reliability of these free thyroid hormone assays using RIA have been thwarted by the anamolous endogenous protein binding found in metabolic disease states.
The development of analog tracers for the measurement of thyroxine (T4) and triodothyroxine (T3) in serum using competitive immunoassay techniques has been subject of much controversy. (Stockigt, et al. The Lancet, Sept. 25, 1982 p. 712; Stockigt, et al. The New England Journal of Medicine Vol. 307, p. 126 (1982); Stockigt, et al. Clinical Endocrinology Vol. 15, 313-318 (1981); Stockigt, et al. Clinical Chemistry 29(7), 1408-1410 (1983); Braun, et al. Clinical Chemistry 29(12), 2057-2060 (1983); Midgley et al. Clinical Endocrinology Vol. 17, 523-528 (1981); Amino et al. Clinical Chemistry 29, 321-325 (1983); and Wilke, Clinical Chemistry 32, 585-592 (1986)).
Designed to measure the amount of free T.sub.3 or T.sub.4 in solution by competitively binding the labelled T.sub.3 or T.sub.4 analogs to a specific antibody thereto, these assays have been found to give misleading results caused by interaction of thyroxine-binding proteins. In familial dysalbuminemic hyperthyroxinemia for example, an abnormal albumin binds up to 50% of the T.sub.4 found in the serum (ca. 12% in normal serum). Because this abnormal albumin binds to the labelled T.sub.4 analog to a greater extent than normal albumin, use of the single-step competitive binding procedure would result in less labelled analog being available with binding sites on the T.sub.4 antibody. This causes an apparent increase in the free T.sub.4 in serum using the one-step procedure.
In patients having severe nonthyroid illness with low levels of T4 and serum prealbumin, the labelled analog does not bind to proteins as well as in normal serum. This causes an apparent decrease in the free T.sub.4 in serum.
Heretofore, methods for measuring free thyroxine hormones in biological fluids using labelled thyroxine analogs have relied on the assumption that the level of antibody-binding of a tracer that does not bind to plasma proteins varies inversely with the concentration of free thyroxine in solution. Commercially available thyroxine analogs have not proven satisfactory tracers in immunoassay because of significant amounts of residual (anamolous) binding to plasma proteins such as thyroxine-binding globulins and albumin.
In accordance with the present invention, we have found thyroxine analogs useful for RIA of thyroxine and triiodothyroxine having a reduced tendency to interact with thyroxine-binding globulin in biological fluids. These new thyroxine analogs are particularly advantageous in measuring thyroxine concentrations in patients having abnormal plasma protein conditions. Moreover, these new analogs are easily iodinatable and controlled giving a high specific activity.